Choi YS, et al. (2010) The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. J Biol Chem 285(23):17754-62
Abstract: Cdc34 is an E2 ubiquitin conjugating enzyme that functions in conjunction with SCF (Skp1/Cullin 1/F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C-terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin K48 and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (F206, Y207, Y210 and Y211) are likely positioned in the vicinity of ubiquitin C-terminal residue V70. Replacement of UBS1 aromatic residues by glycine, or ubiquitin V70 by alanine, decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo as human Cdc34 (1-215), but not Cdc34 (1-200), was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IkappaBalpha ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (F206/Y207, Y210/Y211) in conjugation, with Y210 exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Y210 was required for the transfer of the donor ubiquitin to a receptor lysine on either IkappaBalpha, or a ubiquitin in a manner that depended on the SCF's neddylated RING sub-complex. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C-terminus that contributes to SCF-dependent ubiquitination.
|Status: Published||Type: Journal Article||PubMed ID: 20353940|
Topics addressed in this paper
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|Non-Fungal Related Genes/Proteins|